DNA polymerase are enzymes that synthesize the formation of DNA molecules from deoxynucleotide triphosphates using a template DNA strand and a complementary synthesis primer annealed to a portion of the template. A detailed description of DNA polymerases and their enzymological characterization can be found in Komber, DNA Replication Second Edition, W. H. Freeman (1989).
DNA polymerases have a variety of uses in molecular biology techniques suitable for both research and clinical applications. Foremost among these techniques are DNA sequencing and nucleic acid amplification techniques such as PCR (polymerase chain reaction).
The amino acid sequence of many DNA polymerases have been determined. Sequence comparisons between different DNA polymerase have identified many regions of homology between the different enzymes. X-ray diffraction studies have determined the tertiary structures of Klenow fragment, T7 DNA polymerase, and Taq DNA polymerase. Studies of the tertiary structures of DNA polymerases and amino acid sequence comparisons have revealed numerous structural similarities between diverse DNA polymerases. In general, DNA polymerases have a large cleft that is thought to accommodate the binding of duplex DNA. This cleft is formed by two sets of helices, the first set is referred to as the “fingers” region and the second set of helices is referred to as the “thumb” region. The bottom of the cleft is formed by anti-parallel β sheets and is referred to as the “palm” region. Reviews of DNA polymerase structure can be found in Joyce and Steitz , Ann. Rev. Biochem. 63:777-822 (1994). Computer readable data files describing the three-dimensional structure of some DNA polymerases have been publicly disseminated.
Fluorescently labeled nucleotides have greatly simplified and improved the utility of many procedures in molecular biology. The use of fluorescently labeled nucleotides for labeling polynucleotides in synthesis procedures, has to a large extent replaced the use of radioactive labeling. Fluorescently labeled nucleotides have been widely used in DNA sequencing, see Smith et al Nature 321:674-679 (1986), in PCR, and other forms of polynucleotide fragment analysis.
A major problem with using fluorescently labeled nucleotides is the ability of DNA polymerases to discriminate against the incorporation of fluorescently labeled nucleotides. For example, the inventors have discovered that in competition assays between a TET (6-carboxy-4,7,2′,7′-tetrachlorofluorescein) labeled 2′3′ dideoxynucleotide and the corresponding unlabeled dideoxynucleotide, Taq DNA polymerase incorporates the unlabeled dideoxynucleotide into DNA at least 85 times more frequently than the corresponding unlabeled nucleotide. This discrimination between labeled and unlabeled nucleotides has profound effects on procedures using DNA polymerases to label DNA. For example, much larger amounts of fluorescently labeled nucleotide must be used in sequencing reactions. This large amount of fluorescently labeled nucleotide is expensive and can generate excessive background fluorescence, thereby reducing the yield of sequence information.
In view of the problems arising from the ability of DNA polymerases to discriminate against the incorporation of fluorescently labeled nucleotides, the inventors have developed several novel DNA polymerases that have reduced discrimination against the incorporation of one or more fluorescently labeled nucleotides into DNA.